Native Mass Spectrometry of Membrane Proteins

Keener, James E.; Zhang, Guozhi; Marty, Michael T.

doi:10.1021/acs.analchem.0c04342

Cited by 88 publications

(113 citation statements)

References 177 publications

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“…We first analysed purified recombinant HlgA and HlgB by nMS in order to verify their oligomeric state, in the absence and the presence of detergent micelles as a mimic of the amphiphilic membrane environment. nMS has been gaining ground and has become a key actor in studying membrane protein interactions and dynamics (Allison and Bechara, 2019;Bechara and Robinson, 2015;Calabrese and Radford, 2018;Engen et al, 2020;Keener et al, 2020;Martens and Politis, 2020). It preserves noncovalent interactions and gives information regarding the stoichiometry and binding partners of protein complexes, among which GPCRs (Yen et al, 2017(Yen et al, , 2018.…”

Section: Nms Reveals Hlgb and Hlga Homodimers With Hlgb Being More Prmentioning

confidence: 99%

Mechanisms of GPCR hijacking byStaphylococcus aureus

Leyrat

Lambey

et al. 2021

Preprint

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Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus (SA) bi-component pore-forming leukotoxins to promote bacterial growth and immune evasion. Here we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding to ACKR1 captured by native mass spectrometry (MS). Unexpectedly, Hydrogen/Deuterium exchange-MS revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation of ACKR1-G protein complexes in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR. Our findings may open the way to develop antibiotics inhibiting host receptors binding, a mechanism of action less prone to resistance.

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Section: Nms Reveals Hlgb and Hlga Homodimers With Hlgb Being More Prmentioning

confidence: 99%

Mechanisms of GPCR hijacking byStaphylococcus aureus

Leyrat

Lambey

et al. 2021

Preprint

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“…Native MS uniquely operates under non-denaturing conditions, retaining non-covalent interactions into the gas phase. Native MS can provide unique insight into the composition, architecture and function of large biomolecular assemblies, and has been used to study, among others, ribosomal particles 3 , membrane protein complexes 4 , virus-like particles 5 and endogenous viruses 5–7 (also reviewed in 8–11 ).…”

Section: Introductionmentioning

confidence: 99%

Frequency chasing of individual megadalton ions in an Orbitrap analyzer improves precision of analysis in single molecule mass spectrometry

Woerner

Aizikov

Snijder

et al. 2021

Preprint

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To enhance the performance of charge detection mass spectrometry, we investigated the behavior of macromolecular single ions on their paths towards and within the Orbitrap analyzer. We discovered that ions in mass beyond one megadalton reach a plateau of stability and can be successfully trapped for seconds, travelling a path length of multiple kilometers, thereby enabling precise mass analysis with an effective resolution of greater than 100,000 at m/z 35,000. Through monitoring the frequency of individual ions, we show that these high mass ions, rather than being lost from the trap, can gradually lose residual solvent molecules and, in rare cases, a single elementary charge. Our observations highlight the importance of efficient desolvation for optimal charge detection mass spectrometry and inspired us to implement multiple improved data acquisition strategies. We demonstrate that the frequency drift of single ions due to desolvation and charge stripping can be corrected, which improves the effective ion sampling 23-fold and gives a two-fold improvement in mass precision and resolution, as demonstrated in the analysis of various viral particles.

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“…13 Native mass spectrometry (MS) has emerged as a powerful technique for studying the oligomerization of membrane proteins. [14][15][16] For conventional native MS of membrane proteins, the entire protein-micelle complex is ionized with electrospray ionization (ESI). 14 The detergent adducts are then removed from the protein using collision induced dissociation (CID), and the mass of the bare membrane protein complex reveals the protein stoichiometry and noncovalent ligands that remain bound (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

“…[14][15][16] For conventional native MS of membrane proteins, the entire protein-micelle complex is ionized with electrospray ionization (ESI). 14 The detergent adducts are then removed from the protein using collision induced dissociation (CID), and the mass of the bare membrane protein complex reveals the protein stoichiometry and noncovalent ligands that remain bound (Figure 1). Other membrane mimetics, such as nanodiscs, allow membrane proteins to be solubilized in lipid bilayers during native MS. 14,17,18 Thus, native MS provides rich information and can capture the polydispersity of membrane proteins in different lipid and detergent environments.…”

Section: Introductionmentioning

confidence: 99%

Influenza A M2 Channel Oligomerization is Sensitive to its Chemical Environment

Townsend

Sanders

Rolland

et al. 2021

Preprint

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Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry, ion mobility spectrometry, and size-exclusion chromatography to characterize the oligomeric state of full-length AM2 in a variety of different pH and detergent conditions. Unexpectedly, we discovered that AM2 formed a range of different oligomeric complexes that were strongly influenced by its local chemical environment. The monodisperse tetramer was only observed in select conditions when the antiviral drug, amantadine, was added. Native mass spectrometry of AM2 in lipid nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 forms a wider range of oligomeric states than previously thought possible, which provides new potential mechanisms of influenza pathology and pharmacology.

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Native Mass Spectrometry of Membrane Proteins

Cited by 88 publications

References 177 publications

Mechanisms of GPCR hijacking byStaphylococcus aureus

Mechanisms of GPCR hijacking byStaphylococcus aureus

Frequency chasing of individual megadalton ions in an Orbitrap analyzer improves precision of analysis in single molecule mass spectrometry

Influenza A M2 Channel Oligomerization is Sensitive to its Chemical Environment

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